OBJECTIVE

To evaluate the outcomes of patients with giant cell tumor of bone (GCTB) treated with radiotherapy (RT) with or without surgical resection.

METHODS

We performed a retrospective review of the records from 25 consecutive patients with pathologically confirmed GCTB who had undergone RT between 1956 and 2000.

RESULTS

Patients ranged in age from 11 to 69 years (median <em>3</em>2); 16 were female and 9 were male. The anatomic distribution of lesions was as follows: cervical spine, <em>3</em>; temporal bone, 1; thoracic or lumbar spine, 9; sacrum, 8; ilium, 1, and humerus, radius, and thumb metacarpal, 1 each. Tumors ranged in size from 2 to 20 cm (median 9.5) at their maximal dimension. Thirteen patients had been referred for RT for primary GCTB and 12 had been referred with locally recurrent disease after having undergone one or more other treatments. Fourteen patients had undergone RT for gross disease, and the remaining 11 had been treated with RT after gross total resection. In 10 of these 11 patients, the treatment margins were positive or uncertain. Radiation doses ranged from 25 to 65 Gy (median 46). At a median follow-up of 8.8 years (range 0.67-<em>3</em>4), 7 patients had developed isolated local recurrence, 2 had developed isolated distant recurrence, and <em>3</em> had developed both. The actuarial 5-year overall and disease-free survival rate was 91% and 58%, respectively, and the actuarial 5-year local control and distant metastasis-free survival rate was 62% and 81%, respectively. Univariate analysis suggested that treatment for recurrent disease correlated with a lower disease-free survival rate (8<em>3</em>% vs. <em>3</em><em>3</em>%, p = 0.06), distant metastasis-free survival rate (100% vs. 64%, p = 0.08), and local control rate (8<em>3</em>% vs. 42%, p = 0.08) at 5 years. Of the 12 cases of recurrence, 7 were ultimately successfully treated with additional salvage therapy. In 4 of these patients, salvage therapy included <em>interferon</em>-<em>alpha</em> 2b.

CONCLUSIONS

RT should be considered an adjuvant to surgery or as alternative therapy in cases of GCTB that are unresectable or in which excision would result in substantial functional deficits. When RT is used as primary therapy, the rate of local control seems to be satisfactory. In heavily pretreated patients, however, RT delivered as it was in this series can result in poor local control, and alternative therapies should be considered.

A nonspecific inflammatory and thrombotic reaction termed instant blood-mediated inflammatory reaction (IBMIR) has been reported when allogenic or xenogenic islets come into contact with blood. This reaction is known to cause significant loss of transplanted islets. We hypothesized that IBMIR occurs in patients undergoing total pancreatectomy followed by autologous islet transplantation (TP-AIT) and tested this hypothesis in 24 patients and in an in vitro model. Blood samples drawn during the peritransplant period showed a significant and rapid increase of thrombin-anti-thrombin III complex (TAT) and C-peptide during islet infusion, which persisted for up to <em>3</em> h, along with a decreased platelet count. A concomitant increase in levels of inflammatory proteins IL-6, IL-8 and <em>interferon</em>-inducible protein-10 was observed. An in vitro model composed of pure islets plus autologous blood also demonstrated significantly increased levels of TAT (p<0.05), C-peptide (p<0.05), tumor necrosis factor-<em>alpha</em> (p<0.05) and MCP-1 (p<0.05), as well as strong tissue factor expression in islets. Islet viability decreased significantly but was rescued by the presence of low-molecular-weight dextran sulfate. In conclusion, AIT-induced elevation of TAT and destruction of islets suggests that IBMIR might occur during AIT. Modulating this process may help improve islet engraftment and the insulin independence rate in TP-AIT patients.

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti-Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 5<em>3</em> kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.<em>3</em> (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. <em>Interferon</em>-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U9<em>3</em>7 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-<em>alpha</em> (TNF <em>alpha</em>), and vitamin D<em>3</em> had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D<em>3</em> treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.<em>3</em> identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF <em>alpha</em>, PMA, or vitamin D<em>3</em> had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D<em>3</em> was able to induce the expression of CD14, a marker of mature monocytic phenotype.

To study the local immunological effects of intravesical bacillus Calmette-Guérin (BCG) therapy in superficial bladder cancer patients, the production of interleukin-1 (IL-1), IL-2, IL-6, tumour necrosis factor <em>alpha</em> (TNF <em>alpha</em>), and <em>interferon</em> gamma (IFN gamma) was investigated in the urine. Urine specimens were collected during the six weekly BCG instillations, before instillation, and 2, 4, 6, 8, and 24 h thereafter. Results were standardized to urine creatinine. In general, the concentration of IL-1 increased markedly during the first three BCG instillations, reaching a plateau from instillations <em>3</em> to 6. IL-2 was not detected after the first BCG instillation, but from the second instillation onwards the mean IL-2 concentration increased rapidly. With respect to IL-6, patients had relatively high levels in the urine after the first BCG instillation. A relatively moderate increase of the IL-6 concentration was observed during the following weeks. Like IL-2, TNF <em>alpha</em> was only detected after repeated BCG instillations. Generally the highest TNF levels were found after BCG instillation 5. The presence of IFN gamma could not be demonstrated. With respect to the occurrence of the cytokines during the first 24 h after the BCG instillation, TNF, IL-2, and IL-6 were detectable 2 h after the instillation. In contrast, IL-1 seemed to appear later, i.e. from 4 h onwards. TNF decreased most rapidly; it was nearly absent in 6-h samples. Generally IL-2 was not detectable in the 8-h samples, whereas IL-1 and IL-6 were present up to 8 h after instillation of BCG. The presence of TNF was found less frequently than the presence of IL-1, IL-2, and IL-6. Neutralization experiments indicated that most of the IL-1 present in the urine after BCG treatment was IL-1 <em>alpha</em>. In conclusion, activation of BCG-specific T cells was indicated by the detection of IL-2. The presence of IL-1, IL-6, and TNF <em>alpha</em> might suggest activation of macrophages by intravesically administered BCG, although production by other cell types cannot be excluded. It is suggested that these cytokines, in combination with the leucocytes that are known to be recruited to the bladder in reaction to the BCG treatment, may play an important role in the antitumour activity of BCG against bladder cancer. For monitoring purposes, collection of urine might be performed during the first 6 h after BCG instillations 4-6.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

A role for CXCR3, the receptor for chemokines Mig, IP-10 and interferon-inducible T cell alpha-chemoattractant, in tumour cell migration during melanoma progression has been proposed.

OBJECTIVE

To analyse CXCR3 expression in primary cutaneous malignant melanomas and its comparison with clinicopathological and prognostic factors.

METHODS

A retrospective immunohistochemical study was carried out on formalin-fixed paraffin-wax-embedded sections from 82 patients with primary invasive cutaneous melanomas, with a monoclonal antibody to CXCR3 (clone 49801.111; R&D Systems). Immunoreactivity was semiquantitatively evaluated: labelling intensity (0, absent; 1, weak; 2, moderate; 3, strong) multiplied by the percentage of cells in each of the four intensity categories. A positive staining was considered when the score was >100. Melanomas were categorised by age, sex, primary site, tumour thickness, growth phase, ulceration, lymphocytic infiltration, recurrence, lymph node and distant metastasis, and survival. Univariate and multivariate statistical analyses were carried out.

RESULTS

Of the 82 patients, a positive CXCR3 staining was found in 26 (31.7%) patients, whereas 56 (68.3%) were negative. In univariate analysis, a significant association of CXCR3-positive tumour cell immunostaining with tumour thickness >1 mm (p = 0.003), absence of lymphocytic infiltration (p = 0.04) and the presence of distant metastasis (p = 0.048) was found. Multivariate analysis found tumour thickness as the only independent factor with considerable association with distant metastases.

CONCLUSIONS

Our findings of a positive correlation of CXCR3 tumour cell immunoreactivity in human primary cutaneous melanoma with tumour thickness >1 mm and absence of intratumoral lymphocytic infiltration support the biological implication of CXCR3 in the tumour progression of cutaneous malignant melanoma.

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to <em>interferon</em> (IFN)-<em>alpha</em>/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-<em>alpha</em>/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-<em>alpha</em>/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/beta-catenin signalling pathway contributed to resistance to IFN-<em>alpha</em>/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/beta-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/beta-catenin signalling by glycogen synthesis kinase-<em>3</em> inhibitor (6-bromoindirubin-<em>3</em>'-oxime (BIO)) induced chemoresistance to IFN-<em>alpha</em>/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-<em>alpha</em>/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/beta-catenin signalling pathway induces chemoresistance to IFN-<em>alpha</em>/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.

Silver could prove to be a valuable alternative raw material for antibiotics and disinfectants as it is relatively free of adverse effects. Nano-silver is now been put to practical use in commonly used items, such as, clothes, electric home appliances, and electronic industry, but has not been widely applied in the medical or pharmacological fields. This study was designed to investigate the effects of nano-silver on the production of cytokines by and on the proliferation of peripheral blood mononuclear cells (PBMCs). In addition, we investigated the potential cytotoxic effects of nano-silver on PBMCs. PBMCs from healthy human volunteers were stimulated with 5 mug/ml phytohaemagglutinin (PHA) in the presence of varying concentrations of nano-silver. PBMC proliferations were measured using an aqueous cell proliferation assay kit and supernatants were analyzed using enzyme-linked immunosorbent assays. Interleukin-5 (IL-5), <em>interferon</em>-gamma (INF-gamma), and tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) protein levels were measured to determine the activation state of PBMCs. At levels of over 15 ppm, nano-silver was found to have a significant cytotoxic effect on PBMCs, and PHA-induced cytokine productions were significantly inhibited by nano-silver (IL-5: at 10 ppm, INF-gamma and TNF-<em>alpha</em> at <em>3</em> ppm). Although nano-silver had a cytotoxic effect at high concentration, nano-silver modulated cytokine production in a concentration-dependent manner. These experimental data suggest that nano-silver could be used to treat immunologic and inflammatory diseases.

Leptin is a recently identified hormone produced by the adipocyte ob gene which acts as a negative feedback signal critical to the normal control of food intake and body weight. A number of proinflammatory cytokines, such as interleukin (IL) 1<em>alpha</em>, IL-6, tumor necrosis factor (TNF) <em>alpha</em> and <em>interferon</em> (IFN) gamma, have been proposed as mediators of cancer cachexia. These data suggest that abnormalities in leptin production/release or in its feedback mechanism play a role in cancer patients. To elucidate this we studied the relationship between total serum leptin and serum cytokines IL-1<em>alpha</em>, IL-6, TNF<em>alpha</em> as well as the production of leptin and cytokines by peripheral blood mononuclear cells (PBMC) isolated from cancer patients. Sixteen advanced cancer patients (mainly stage IV) with tumors at different sites were included in the study. The serum levels of leptin in cancer patients were significantly lower than those of healthy individuals at all times (7 a.m., noon, <em>3</em> p.m.). No significant differences were found in circadian rhythm between patients and controls. Serum levels of IL-1<em>alpha</em>, IL-6, and TNF<em>alpha</em> were significantly higher in cancer patients than in healthy individuals. An inverse correlation between serum levels of leptin and IL-6 was found in cancer patients. The production in culture of leptin by unstimulated PBMCs and those stimulated by phytohemagglutinin M or by phorbol myristate acetate isolated from cancer patients was very low; no differences were observed in comparison with leptin production by PBMCs from healthy individuals.

We have recently observed that the selective adenosine A<em>3</em> receptor agonist N6-(<em>3</em>-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) augments interleukin-10 and inhibits tumor necrosis factor-<em>alpha</em> production in endotoxemic mice. In the present study, we extended our investigations into the effect of this compound on the bacterial lipopolysaccharide (endotoxin)-induced inflammatory response in the BALB/c, as well as in the C57BL/6 interleukin-10+/+ and the interleukin-10 deficient C57BL/6 interleukin-10(0)/0 mice strains. In the BALB/c mice, i.p. pre-treatment with IB-MECA (0.2 and 0.5 mg/kg) decreased lipopolysaccharide (60 mg/kg i.p.)-induced plasma levels of interleukin-12 (p40 and p70), <em>interferon</em>-gamma, and nitrite/nitrate (breakdown products of nitric oxide (NO)). On the other hand, pre-treatment with this compound failed to influence lipopolysaccharide-induced plasma interleukin-1 <em>alpha</em>, interleukin-6, and corticosterone concentrations. Similar to its effect in BALB/c mice, IB-MECA enhanced the release of interleukin-10 in the C57BL/6 interleukin-10+/+ mice. Furthermore, IB-MECA inhibited the production of interleukin-12, <em>interferon</em>-gamma, and NO in both the C57BL/6 interleukin-10+/+ and C57BL/6 interleukin-10(0)/0 mice, suggesting that the inhibition of pro-inflammatory cytokine production by this compound is independent of the increased release of interleukin-10. Finally, pre-treatment with this compound protected mice against lipopolysaccharide (60 mg/kg i.p.)-induced lethality. These results indicate that stimulation of adenosine A<em>3</em> receptors has potent anti-inflammatory effects and may represent a potential strategy in the treatment of septic shock and other inflammatory diseases.

OBJECTIVE

Recombinant human interferon-alpha (r-IFN-alpha) is often successfully used in the treatment of patients with chronic viral hepatitis B and C. Thyroid dysfunction has been reported to occur with variable frequency during r-IFN-alpha therapy especially in patients with preexisting thyroid autoimmunity. We have prospectively evaluated the effect of r-IFN-alpha on various aspects of thyroid function in patients with HCV chronic hepatitis.

METHODS

Thirty-two patients with HCV chronic active hepatitis were studied prospectively before and during r-IFN-alpha therapy. Serum TSH, FT4, FT3, and thyroid receptor (TSR) and thyroid peroxidase (TPO) antibodies, and the iodide-perchlorate discharge test (I-C10(4)) to detect subtle defects in the thyroid organification of iodide were carried out during the study. Thyroid radioactive iodine uptakes (RAIU) were obtained in patients who developed thyrotoxicosis.

RESULTS

All patients were clinically and biochemically euthyroid prior to r-IFN-alpha therapy with negative I-C10(4) discharge tests. Four patients became thyrotoxic, 3 secondary to destructive or inflammatory thyroiditis with a low thyroid RAIU, and 1 patient developed hypothyroidism. The I-C10(4) discharge test became positive in 7 of the 32 patients studied prospectively; 5 of these patients did not develop other evidence of thyroid dysfunction and did not have positive TPO antibodies. In these 5 patients the test became negative after r-IFN-alpha was discontinued. Appropriate therapy of the patients with thyrotoxicosis (methylprednisolone for 3 patients with destructive thyroiditis and methimazole for 1 patient with hyperthyroidism) or with hypothyroidism (L-thyroxine) was successful.

CONCLUSIONS

Thyroid dysfunction, especially destructive or silent thyroiditis resulting in thyrotoxicosis, is not infrequently observed in patients receiving r-IFN-alpha therapy for chronic active hepatitis. Although underlying autoimmune thyroid disease appears to predispose patients to develop thyroid dysfunction, other patients become thyrotoxic or hypothyroid in the absence of baseline positive TPO-Ab. Subtle defects in the thyroidal organification of iodine as determined by the I-C10(4) discharge test, in the absence of autoimmune thyroid disease, was observed in 5 patients who remained euthyroid, suggesting that r-IFN-alpha directly reduces the intrathyroidal organification of iodine.

Tissue factor is a cell-surface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury by interacting with blood clotting factor VII/VIIa and which is implicated in various pathological processes. When bound to tissue factor, factor VII is readily converted to the active protease factor VIIa by trace amounts of factors Xa, IXa or VIIa. Human tissue factor consists of 26<em>3</em> residues, the first 219 of which comprise the extracellular region. We have determined the crystal structure of the extracellular region at a resolution of 2.2 A. Tissue factor consists of two immunoglobulin-like domains associated through an extensive, novel, interdomain interface region. The binding site for factor VII lies at the interface region and involves residues from domain 1 and an extended loop (binding 'finger') of domain 2. This is the first reported structure of a representative of the class 2 cytokine receptor family, which also includes <em>interferon</em>-<em>alpha</em>, <em>interferon</em>-gamma (refs 2, <em>3</em>) and interleukin-10 (ref. 4) receptors.

Type I <em>interferons</em> (IFNs) derived from plasmacytoid dendritic cells (PDCs) are critical for antiviral responses; however, the mechanisms underlying their production remain unclear. We have identified a receptor, PDC-TREM, which is associated with Plexin-A1 (PlxnA1) on the PDC cell surface and is preferentially expressed after TLR-stimulation. Limited TLR signals induced PDC-TREM expression but failed to induce IFN-<em>alpha</em> production. However, when coupled with Sema6D, a ligand for Plexin-A1, limited TLR-stimulation resulted in PDC-TREM-mediated DAP12-dependent phosphorylation of phosphoinositide <em>3</em>-kinase (PI<em>3</em>K) and extracellular regulated kinase (Erk) 1/2 at 6-9 h, and IFN-<em>alpha</em> was produced. Inhibition of PDC-TREM expression by pdctrem-shRNA, blocking of PDC-TREM-binding with PlxnA1 by PDC-TREM mAb, and DAP12 deficiency all resulted in greatly reduced PDC-TREM-dependent activation of signaling molecules and IFN-<em>alpha</em> production. Thus, PDC-TREM is responsible for IFN-<em>alpha</em> production, whereas TLR signals are essential for PDC-TREM expression.

OBJECTIVE

There is evidence that sleep facilitates the adaptive immune response to infectious agents and, thereby, supports immunologic memory. The effect might be attained by sleep-induced changes in the number and function of dendritic cells (DCs), which play a key role in the initiation of the immune response. This study aimed to dissociate effects of sleep and circadian rhythm on circulating numbers of DC precursors, ie, CD14+CD16- and CD14(dim)CD16+ monocytes, myeloid dendritic cell precursors (pre-mDC), and plasmacytoid dendritic cells (PDC) and on 2 key cytokines produced by these cells, ie, interleukin (IL)-12 and interferon (IFN)-alpha.

METHODS

In a within-subject cross-over design, human subjects were examined on 2 occasions, ie, during a normal sleep-wake cycle and during 24 hours of wakefulness. Blood was sampled every 1.5 hours during nighttime and every 3 hours during daytime.

METHODS

Experiments took place under controlled laboratory conditions.

METHODS

Twenty-seven healthy men aged between 18 and 30 years.

RESULTS

Compared with wakefulness, sleep was associated with a striking increase in the number of pre-mDC producing IL-12, which is a main inducer of Th1 responses. In addition, sleep slightly decreased PDC and also T cell counts but did not affect IFN-alpha production by PDC. Sleep, however, substantially decreased numbers of CD14(dim)CD16+ monocytes, probably reflecting increased margination of the cells upon a sleep-related drop in catecholamine release.

CONCLUSIONS

Our data identify pre-mDC producing IL-12 as a basic target of sleep that is most closely related to mature APC function and whereby sleep can effectively enhance adaptive immune responses.

BACKGROUND

Hepatocellular carcinoma (HCC) recurrence after ablation therapy for primary tumors is common.

METHODS

To evaluate the effectiveness of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) in preventing HCC recurrence, <em>3</em>0 eligible patients were randomized into three groups: 11 patients treated with three mega units (MU) of IFN-<em>alpha</em> three times weekly for 24 months (IFN-<em>alpha</em>-continuous group), 9 patients treated with <em>3</em> MU of IFN-<em>alpha</em> daily for 10 days every month for 6 months followed by <em>3</em> MU of IFN-<em>alpha</em> daily for 10 days every <em>3</em> months for a further 18 months (IFN-<em>alpha</em>-intermittent group), and 10 patients who received no IFN-<em>alpha</em> therapy (control group). The three groups were comparable in terms of etiology, demographics, and laboratory data at entry and HCC characteristics.

RESULTS

After a median follow-up of 27 months (range 4-5<em>3</em> months), 9 patients (90%) in the control group and 9 patients (45%) in 2 treatment groups (6 patients in the IFN-<em>alpha</em>-continuous group and <em>3</em> patients in the IFN-<em>alpha</em>-intermittent group) developed an HCC recurrence (P = 0.021). Cumulative HCC recurrence rates in the IFN-<em>alpha</em>-intermittent, IFN-<em>alpha</em>-continuous, and control groups were 22.2%, 27.<em>3</em>%, and 40% at the end of 1 year and <em>3</em><em>3</em>.<em>3</em>%, 54.6%, and 90% at the end of 4 years (P = 0.0<em>3</em>75), respectively (control vs. IFN-<em>alpha</em>-intermittent group, P = 0.012<em>3</em>; vs. IFN-<em>alpha</em>-continuous group, P = 0.0822). If both IFN-<em>alpha</em> groups were combined, the cumulative HCC recurrence rate of the patients treated with IFN-<em>alpha</em> and the control group was 25% and 40% at the end of 1 year and 47% and 90% at the end of 4 years, respectively (P = 0.01<em>3</em>5).

CONCLUSIONS

The data suggested that IFN-alpha therapy may reduce HCC recurrence after medical ablation therapy for primary tumors.

OBJECTIVE

We provide a current review of the management of advanced renal cell carcinoma.

METHODS

A comprehensive literature review of peer reviewed articles which address the current management of metastatic renal cell carcinoma was performed.

RESULTS

Renal cell carcinoma is the seventh leading cause of cancer, accounting for <em>3</em>% of malignancies in men. The incidence of renal cell carcinoma has increased significantly by <em>3</em>8% from 1974 through 1990 at least in part related to earlier diagnosis with the common use of new radiological techniques. Cytotoxic chemotherapy remains poor as a treatment alternative. <em>Interferon</em>-<em>alpha</em> produces responses in 15 to 20% of patients but clinical usefulness as monotherapy has been surpassed by interleukin-2 (IL-2). IL-2 is the first immunotherapy to produce durable remissions resulting in approval by the Food and Drug Administration. Although high dose bolus IL-2 schedules have the longest followup, IL-2 administered on other schedules may have enhanced efficacy. Randomized trials are attempting to delineate the appropriate role for various doses and schedules.

CONCLUSIONS

Advanced renal cell carcinoma, once a disease relegated to the incurable, during the last decade has evolved into a malignancy that may be associated with cure. The first evidence of this potential is the clear and unequivocal demonstration that IL-2 produces durable complete remissions. Building upon this immunotherapeutic approach the future treatment of renal cell carcinoma will incorporate new immunological technology, including gene, dendritic cell, vaccine and antibody therapy.

Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I <em>interferon</em> (IFN-<em>alpha</em>/beta). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor <em>3</em> null (IRF<em>3</em>(-/-)) mice, indicating that IFN production was independent of the IRF<em>3</em> pathways to IFN gene transcription, consistent with the IFN type produced (predominantly <em>alpha</em>).

Ribavirin (RBV), used in combination with <em>alpha</em> <em>interferon</em> to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [(<em>3</em>)H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.

To examine the effects of <em>interferon</em> (IFN) therapy on clinical, biochemical, and histological features in patients with compensated hepatitis C virus (HCV)-related cirrhosis, we have conducted a randomized, controlled trial of IFN therapy versus observation. Eight centers included a total of 99 patients with biopsy-proven cirrhosis. IFN-<em>alpha</em>2b, <em>3</em> million units three times per week, or no antiviral therapy was given for 48 weeks. Twenty-three patients dropped out. End-of-treatment biochemical response was not observed in any of the <em>3</em>9 controls but was observed in 6 of the 47 treated patients (P <.02); sustained biochemical response was obtained in only 2 treated patients. Controls and treated patients did not significantly differ with regard to the changes in serum level of albumin, bilirubin, <em>alpha</em>-fetoprotein, in plasma prothrombin, in histological activity, or liver collagen content. During trial or follow-up (160 +/- 57 weeks), hepatocellular carcinoma developed in 9 controls and 5 treated patients (NS); decompensation of cirrhosis occurred in 5 controls and 7 treated patients. Seven controls and 10 treated patients died. In conclusion, in patients with compensated HCV-related cirrhosis, a 48-week course of IFN therapy is safe and is able to induce end-of-treatment biochemical response in a significant proportion of patients. However, a 48-week course of IFN therapy usually fails to achieve sustained response and, within the limit of this study, did not significantly improve the <em>3</em>-year outcome. Therefore, a longer course of IFN therapy or combination therapy with ribavirin should be evaluated in patients with HCV-related cirrhosis.

OBJECTIVE

We undertook this study to determine the activity and tolerability of sorafenib administered with interferon alfa-2b (IFN-alpha-2b) as first- or second-line therapy in metastatic renal cell cancer (RCC).

METHODS

Between November 2004 and October 2006, 40 patients at two sites were enrolled onto a phase II trial of sorafenib plus IFN-alpha-2b. Treatment consisted of 8-week cycles of sorafenib 400 mg orally bid plus IFN-alpha-2b 10 million U subcutaneously three times a week followed by a 2-week break. Patients were eligible to receive additional cycles of therapy until disease progression. Dose reduction of both drugs by 50% was permitted once for toxicity.

RESULTS

The response rate was 33% (95% CI, 19% to 49%; 13 of 40 patients), including 28% partial responses (n = 11) and 5% complete responses (n = 2). Responses were seen in treatment-naïve and interleukin-2 (IL-2) -treated patients within the first two cycles. The median duration of response was 12 months. With a median follow-up time of 14 months, median progression-free survival time was 10 months (95% CI, 8 to 18 months), and median overall survival time has not yet been reached. Fatigue, anorexia, anemia, diarrhea, hypophosphatemia, rash, nausea, and weight loss were the most common toxicities. Grade 3 toxicities were uncommon but included hypophosphatemia, neutropenia, rash, fatigue, and anemia. Dose reductions were required in 65% of patients.

CONCLUSIONS

The combination of sorafenib and IFN-alpha-2b has substantial activity in treatment-naïve and IL-2-treated patients with RCC. The toxicity exceeded that of either drug alone, but dose reductions and breaks between cycles allowed for chronic therapy. A larger, randomized trial would determine whether there is any advantage to this regimen compared with sorafenib alone.

Conventional therapies for primary chronic cold agglutinin disease (CAD) are ineffective, but remissions after treatment with the anti-CD20 antibody rituximab have been described in a small, prospective trial and in some case reports. In this study we report on <em>3</em>7 courses of rituximab administered prospectively to 27 patients. Fourteen of 27 patients responded to their first course of rituximab, and 6 of 10 responded to re-treatment. In both groups combined, responses were achieved after 20 of <em>3</em>7 courses, giving an overall response rate of 54%. We observed 1 complete and 19 partial responses. Two nonresponders and <em>3</em> patients who experienced relapse received second-line therapy with <em>interferon</em>-<em>alpha</em> combined with a new course of rituximab, and 1 nonresponder and 2 patients who experienced relapse achieved partial responses. Responders achieved a median increase in hemoglobin levels of 40 g/L (4 g/dL). Median time to response was 1.5 months, and median observed response duration was 11 months. We conclude that rituximab is an effective and well-tolerated therapy for CAD. Histologic and flow cytometric findings suggest that some of the effect may be mediated by mechanisms other than the elimination of clonal lymphocytes. We were unable to predict responses from the hematologic, immunologic, or histologic parameters before therapy.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a pivotal role in several immunoinflammatory and autoimmune diseases. In this study we examined the role of MIF in the development of immunoinflammatory diabetes induced in susceptible strains of mice by multiple low doses of streptozotocin. We found that MIF protein was significantly elevated in islet cells during the development of diabetes, and that targeting MIF activity with either neutralizing antibody or the pharmacological inhibitor (S,R)-<em>3</em>-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, markedly reduced clinical and histopathological features of the disease, such as hyperglycemia and insulitis. Lymphocytes from mice treated with the MIF inhibitors exhibited reduction of both islet antigen-specific proliferative responses and adhesive cell-cell interactions. Neutralization of MIF also down-regulated the ex vivo secretion of the proinflammatory mediators, TNF-<em>alpha</em>, <em>interferon</em>-gamma, and nitric oxide, while augmenting that of the antiinflammatory cytokine, IL-10. This study provides the first in vivo evidence for a critical role for MIF in the immune-mediated beta-cell destruction in an animal model of human type 1 diabetes mellitus and identifies a new therapeutic strategy for the prevention and treatment of this disease in humans that is based on the selective inhibition of MIF activity.

Previous studies have shown that human cytomegalovirus (CMV) is a potent elicitor of <em>interferon</em>-stimulated gene (ISG) expression. Induction of the <em>interferon</em> pathway does not require replication-competent virus, and envelope glycoprotein B (gB) from CMV is a viral structural component that can directly induce transcription of ISGs. Here we extend these earlier findings by defining the consequences of inducing the <em>interferon</em> pathway. We found that cells respond to CMV or soluble gB by establishing a functional antiviral state within cell types critical in CMV biology, such as fibroblasts and endothelial cells. We have also discovered new insights into the mechanism by which the pathway is initiated. <em>Interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>), a key transcriptional regulator of cellular <em>interferon</em> responses, is activated by CMV virions and soluble gB. Thus, IRF<em>3</em> becomes activated via "outside-in" signal transduction events. This is a novel mechanism of activation of this key transcription factor by viruses. In comparison to soluble gB (gB(1-750)), which comprises the entire ectodomain of gB, a truncation mutant encompassing only the amino-terminal region of gB (gB(1-460)) was markedly less effective at inducing antiviral responses. This indicates that the region of gB from residues 461 to 750 is important for initiation of the antiviral response. In addition, CMV and gB establish an antiviral state in <em>alpha</em>/beta <em>interferon</em> null cells, illustrating that primary induction of ISGs by CMV and gB is sufficient to establish the antiviral response and that <em>interferon</em> secretion is not necessary for the antiviral effect. Taken together, our findings reveal that CMV initiates a coordinated antiviral response through contact between gB and an as-yet-unidentified cell surface receptor(s).

The induction and inhibition of the <em>interferon</em> (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to <em>alpha</em>/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-<em>alpha</em>/beta or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-beta induction pathway have been analyzed. The transcription factor NF-kappa B was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-kappa B by either SFV or tumor necrosis factor <em>alpha</em>. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. <em>Interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-<em>3</em> was not seen in nuclear extracts. In contrast, an IRF-<em>3</em>-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-<em>3</em> function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.

OBJECTIVE

To examine and compare the molecular and cellular processes leading to radiation fibrosis and pneumonitis in C57BL/6J and C<em>3</em>H/HeN mice.

METHODS

At indicated times after various doses of thoracic irradiation, the cell populations obtained by bronchoalveolar lavage of C57BL/6J mice were differentially analyzed by cytology and assessed by RNase protection (RPA) assay for levels of cytokines and related genes. The molecular responses in bronchial alveolar lavage (BAL) populations were compared with those in whole lung of C57BL/6J mice and with those of C<em>3</em>H/HeN mice. The former strain develops late radiation fibrosis, whereas the latter develop subacute radiation pneumonitis.

RESULTS

In C57BL/6J mice, a decrease in the total number of BAL cells was found 1 week after 6, 12, or 20 Gy thoracic irradiation with a subsequent dose-dependent increase up to 6 months. After 12 and 20 Gy, large, foamy macrophages and multinucleated cells became evident in BAL at <em>3</em> weeks, only to disappear at 4 months and reappear at 6 months. This biphasic response was mirrored by changes expression of mRNA for proinflammatory cytokines and the Mac-1 macrophage-associated antigen. As with BAL, whole lung tissue also showed biphasic cytokine and Mac-1 mRNA responses, but there were striking temporal differences between the two compartments, with changes in whole lung tissue correlating better than BAL with the onset of fibrosis in this strain. The radiation-induced proinflammatory mRNA responses had strain-dependent and strain-independent components. Thoracic irradiation of C<em>3</em>H/HeN induced similar increases in tumor necrosis factor (TNF)-<em>alpha</em>, interleukin (IL)-1<em>alpha</em>/beta, and <em>interferon</em> (IFN)-gamma mRNA expression in lung as it did in C57BL/6J mice during the "presymptom" phase at 1-2 months. However, immediately preceding and during the pneumonitic time period at <em>3</em>-4 months, TNF-<em>alpha</em> and IL-1<em>alpha</em>/beta mRNAs were highly upregulated in C<em>3</em>H/HeN mice, which develop pneumonitis, but not in C57BL/6J mice, which do not. At the onset of radiation fibrosis in C57BL/6J mice (5-6 months), irradiated lungs had increased levels of IL-1<em>alpha</em>/beta and IFN-gamma mRNA expression, but the TNF-<em>alpha</em> response was, notably, still muted.

CONCLUSIONS

The major molecular and cellular events in lungs of C57BL/6J and C<em>3</em>H/HeN mice, which develop late fibrosis and subacute pneumonitis after thoracic irradiation respectively, take place within the interstitium and are not reflected within BAL populations. The initial proinflammatory responses are similar in the two strains, but later responses reflect the latent time to lesion development. TNF-<em>alpha</em> expression at <em>3</em>-4 months may be important in radiation-induced pneumonitis, and its downregulation is important in avoiding this radiation-induced complication.